The selection of reference substances for improving chemical persistence assessment in higher tier OECD 309 simulation tests

The reliable assessment of chemical persistence is critical for environmental risk evaluation, yet variability in OECD 309 test guideline (TG) for surface water simulation tests can undermine confidence in results. Current reference substances, aniline and sodium benzoate, degrade rapidly and may not adequately reflect inoculum performance, limiting their value for test validation. This study applied a structured, multi-step process to identify and validate alternative reference substances that improve robustness in OECD 309 testing. From an initial list of 32 candidates, eight screened for physicochemical suitability and biodegradation behaviour using an adapted OECD 301D TG method. Two substances, caffeine and 2,4-dichlorophenoxyacetic acid (2,4-D), were selected for further evaluation under OECD 309 TG conditions at two temperatures (12 and 20°C) and two concentrations (1 and 10 µg L^-1). Caffeine demonstrated slower but consistent mineralisation compared to existing references, making it a suitable positive control for inoculum viability. 2,4-D exhibited limited degradation, particularly at 12°C, supporting its use as a negative benchmark for persistence. Adoption of these substances could strengthen OECD TG 309 validity criteria and improve regulatory persistence assessments under frameworks such as REACH. These findings provide a basis for an interlaboratory ring test to confirm their suitability and enhance comparability of persistence data.